Stromal cells affect prostate cancer (PCa) cells and are themselves altered by interactions with PCa cells during all phases of invasion and metastasis, ultimately leading to PCa colonization of bone. Interactions among the cells present in the metastatic PCa niche, including bone, are the focus of this project, with emphasis on the heparan sulfate proteoglycan perlecan, Pin, molecules that bind to heparan sulfate (HS), and receptors for HS binding proteins which support PCa growth and progression. We hypothesize that both increases in Pin expression in the reactive stroma and Pin degradation by the PCa cells support aggressive growth of invasive PCa cells. To precisely define the structural and functional roles that Pin, its various domains and its associated HS chains play in skeletal metastasis of prostate cancer, we will use a combination of biochemical approaches, in vitro models, mouse models and clinical specimens to address the following specific aims: 1) Use 2-D and 3-D cell culture, animal models and clinical specimens to examine the molecular mechanisms leading to increases in Pin gene transcription and expression by reactive stromal cells, bone marrow stromal cells, and by PCa cells undergoing transdifferentiation in bone. Use the information to evaluate pathway inhibitors for the ability to reduce Pin expression and its associated signaling networks that alter gene expression, support PCa growth and favor progression to colonization of bone. 2) Examine the degradation of Pin by catabolic enzymes activated by invasive PCa cells in the metastatic niche and to identify the functional changes which occur by a.) loss of the extracellular scaffolding function of intact Pin and b.) gain of growth, angiogenic and transdifferentiating functions owed to bioactive Pin -derived proteolytic fragments and binding molecules released by catabolism of HS chains. 3) Evaluate the usefulness of antibodies recognizing neoepitopes created by Pin degradation as new sensitive diagnostics for detection of invasive PCa cells growing in bone, first in pre-clinical animal models, then in specimens from PCa patients. Together, these interwoven aims will allow us to evaluate the link between Pin expression, catabolism, activation and HS-dependent growth and progression of PCa ultimately to the bone metastatic niche. RELEVANCE (See instructions): Recent data support the paradigm shifting conclusion that changes in the bone marrow stromal cells surrounding prostate cancer cells growing in bone provide two translational opportunities for treating PCa, the ability to: 1) follow changes in reactive stroma as early indicators of tumor growth and invasion at secondary sites; and 2) target pathways activated in reactive stroma as novel interventions of cancer growth and disease progression after an invasive phenotype has been established. We will combine basic and translational approaches to achieve these dual goals.